Conventional vaccines are based on microbial germs which are either dead or have been attenuated by various physico-chemical treatments. Although effective, these vaccines do not have the same antigenic power as the living germs, with the result that there is a considerable loss of antigenic activity during preparation of the vaccines.
It is known that the antigenic power of certain germs is associated with the ribosomes of the microbial cells, and that the antigenic activity of these ribosomal fractions is only developed in the presence of immunity adjuvants. These immunity adjuvants are present in the natural state in microbial cell membranes in the form of molecules of the peptidoglycan type.
The vaccine of this invention is an acellular bacterial vaccine comprising:
as antigenic material, the ribosomes extracted from the bacterias against which a protection is wanted, and
as immunity adjuvant, the peptidoglycans extracted only from the cell membranes of at least one of the bacterias previously used.
The terms ribosomes and ribosomal fraction will be used synonymously and designate the ribosomes per se of bacteria which are a well known part of the bacteria, which may be contaminated by a minor part of other materials because it is well known in this field that purity of 100% cannot be reached especially on industrial scale.
The ratio between the ribosomal fraction and the membranous peptidoglycans may vary according to the nature of the bacterias used but is desirably comprised between 1/5 to 5/1 in weight and is especially 1/1,5 of ribosomal fraction/membranous fraction in weight.
The best ratio may be easily determined by methods well known in biology such as the Ouchterlany method which shows the best antibodies response by checking the precipitation zones corresponding to the presence in for example rabbit serum (treated with the vaccine) of specific antibodies desired.
The vaccines according to the present invention may be used as it is well known for other vaccines including dosage and number of unit dosage by day or by month.
The dosage of each unit dose may vary widely according to the bacterias used.
The vaccines of the present invention are particularly useful in the prevention and treatment of several conditions in the sphere of the oto-rhino-laryngology for example against rhinitis, sinusitis, pharyngitis, rhino-tracheobronchitis, asthma and otitis-pharyngitis. In this case the vaccines contain as active ingredients for example:
the ribosomal fraction of
Klebsiella pneumoniae PA1 Diplococcus pneumoniae PA1 Streptococcus pyogenes PA1 Hemophilus influenzae, and PA1 Klebsiella pneumoniae.
the membranal glycopeptides of
This kind of vaccine may be used especially under aerosol or injectable form. In this case each unit dose contains from 0.005 to 0.030 mg of ribosomal fraction and from 0.010 to 0.060 mg of membranal glycopeptides, preferably 0.020 mg and 0.030 mg respectively.
In one of the specific embodiment of the invention the vaccines for prevention and treatment of ORL disease contain as active ingredients:
______________________________________ the ribosomal fraction of ______________________________________ Klebsiella pneumoniae 30 to 40 parts in weight Diplococcus pneumoniae 25 to 35 parts in weight Streptococcus pyogenes 25 to 35 parts in weight Hemophilus influenzae 1 to 10 parts in weight, ______________________________________
and
membranal glycopeptides of Klebsiella pneumoniae in a ratio comprised between 2/1 and 1/2 in weight.
The present invention also concerns a process for preparing the vaccines stated above in which the ribosomal fraction is prepared by
(a) cultivating the strain of bacterias corresponding to the ribosomal fraction desired on a growth medium,
(b) decantating the bacterias cells,
(c) grinding the bacterias cells in a buffer solution and eliminating the undestroyed bacterias to obtain an homogeneous cell macerate,
(d) sedimenting the cells component other than ribosomal fraction,
(e) sedimenting from the supernatant solution obtained the ribosomal fraction,
(f) washing the ribosomal fraction with a solution containing sodium dodecyl sulphate to eliminate the protein
(g) sedimenting the washed ribosomal fraction; in which the membranal glycopeptides are prepared by
(a') cultivating the strain of bacterias corresponding to the ribosomal fraction desired on a growth medium,
(b') decantating the bacterias cells,
(c') grinding the bacterias cells in a buffer solution and eliminating the undestroyed bacterias to obtain an homogeneous macerate,
(d') sedimenting the membranal fraction from the homogeneous macerate,
(e') suspending the membranal fraction in saline solution and heating the suspension to destroy lytic enzyme,
(f') centrifugating the suspension and washing at least one time the residue containing the membrane with saline solution containing at least one salt among NaCl and MgCl.sub.2,
(g') collecting the membrane and digesting the membrane,
(h') sedimenting the glycopeptides thus obtained, mixing these glycopeptides and the ribosomal fraction of
The bacterias are cultivated by any of the methods normally used in microbiology. So far as the types of germs treated are concerned, they will of course be governed by the required vaccine and may be determined by known method.
The cells are decanted from the growth medium in (b) by low speed centrifugation or by filtration for example on a Westfalia clarifier.
The buffer used in step (c) may be for example a tris-MgCl.sub.2 -NaCl buffer (pH 7) that is it contains
HCl: 0,01 M PA0 NaCl: 9 g/l PA0 MgCl.sub.2,: 6H.sub.2 O 0,01 M.
The homogeneous macerate of bacterias obtained in step (c) may be obtained by any known grinding method, although it is preferably obtained by subjecting a culture of germs to ultrasonic treatment at low temperature in order to macerate the germs, after which the crude macerate is centrifuged so as to sediment and eliminate the germs which have not been destroyed, this centrifuging step preferably being carried out at 10.sup.4 g. The supernatant phase thus obtained constitutes the homogeneous macerate used in step (d).
But if the quantity of cells is too important it is possible to grind the cell with a Manton-Gaulin grinder instead of a ultrasonic treatment.
To sediment the cell components other than ribosomal fraction in (d) it is possible to centrifuge the homogeneous cell macerate. This centrifuging step (step d) is carried out under an acceleration of from 2.times.10.sup.4 to 6.times.10.sup.4 g and preferably under an acceleration of 4.times.10.sup.4 g over a period of about 20 minutes or 3.times.10.sup.4 g over 45 minutes.
Sedimentation of step (e) may be conducted by centrifugating the solution of step (c) at very high speed, 10.sup.5 to 2.times.10.sup.5 g and preferably around 140,000 g for 3 hours, or it is possible to precipitate the ribosomal fraction with ethanol 95% at -20.degree. C. with addition of polyethylenglycol 4000 at 100 g/l.
In this case the precipitate may be recovered by any known method such as filtration or low speed centrifugation.
The ribosomal fraction of step (e) is washed with a solvent of protein which is sodium dodecyl sulphate (SDS). This solvent may be for example the tris-HCl-MgCl.sub.2 -NaCl buffer cited above containing in addition 0.5% of (SDS).
After washing the ribosomal fraction is sedimented in step (g) by the same process as used in step (e).
The steps (a'), (b'), (c') and (d') may be conducted in the same manner as described for steps (a), (b), (c) and (d). The main component of cell component sedimented in step (d) is the membranes.
Extraction of the membranes from the centrifuging residue of step (d) is preferably preceded by a stage in which the lytic enzymes of the cell components present in the residue are destroyed, for example by heating the residue to 100.degree. C., optionally after redissolution.
The actual extraction of the membranes from the centrifuging residue of step (d) is preferably carried out by treating the cell components of the residue, optionally after destruction of the lytic enzymes, with a saline solution, for example 1 M sodium chloride or a tris-HCl-NaCl-MgCl.sub.2 buffer as cited above, either once or several times and centrifuging the suspension obtained. The supernatant phase left after this centrifuging step, which is eliminated, contains the non-membranal impurities such as proteins and nucleic acids, whilst the residue contains the membranes. This centrifuging step is preferably carried out at 2.times.10.sup.4 g.
Following separation of the saline solution containing the impurities, such as proteins and nucleic acids, the membranes are digested in step (g') in the presence of proteolytic enzymes, preferably trypsin and chymotrypsin, in solution at pH 8 for 4 hours at a temperature of 37.degree. C.
After digestion, homogenisation is completed by subjecting the solid digestion fraction to further ultrasonic maceration. The product thus obtained constitutes the peptidoglycanic fraction of the vaccine.
Thereafter it is sufficient to mix the ribosomal fraction obtained and the membranal peptidoglycanes in order to obtain a vaccine according to the invention.
When the ribosomal fraction or peptidoglycans used come from different strains of bacterias each strain is preferably treated independently but it is possible to treat all strains together in the process.
The active parts of the vaccine according to the invention are mixed for administration with suitable pharmaceutical carriers which are usual in the art.